This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: An aliquot of each sample (~400 [unreadable]g) was lyophilized for an experiment to determine the proportion of 2,3-linked to 2,6-linked sialic acids.. The dried samples were dissolved with 0.1 M Tris-HCl buffer (pH ~8.0) and heated at 100[unreadable]C for 5 min to denature the protein. After cooling to room temperature, the samples were treated with trypsin and chymotypsin and incubated at 37oC overnight. Each of the tryptic-chymotryptic digests was passed through a C18 sep pak cartridge, cleaned with 5% acetic acid, and glycopeptides/peptides were eluted subsequently in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid and 100% isopropanol. The glycopeptide eluates were dried initially under a stream of nitrogen gas to evaporate the isopropanol and eventually lyophilized. The glycopeptides were first treated with a neuraminidase that is specific to cleave 2,3-linked sialic acids and digestion was undertaken at 37oC for exactly 1 hr. Each of the digests was passed through a C18 column thereafter and the 2,3-linked sialic acids were eluted with 5% acetic acid and lyophilized. The residual glycopeptides were eluted in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid, and 100% isopropanol. The latter eluates were evaporated of isopropanol and eventually lyophilized. The residual glycopeptides were finally treated with a general neuraminidase and incubated at 37oC overnight to cleave 2,6-linked sialic acids. At the end of the second neuraminidase treatment, the digests were dried under a stream of nitrogen gas. All digests were dissolved with nanopure H2O, sonicated in ice for 5 min and transferred into injection vials for sialic acids analysis. A mix of sialic acids standards with known number of moles was prepared and diluted serially into four concentrations to establish a calibration equation. The number of moles of each residue in the sample was quantified by linear interpolation from the calibration equation. The sialic acids were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The individual sialic acids were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient program used the following mobile phase eluents: 100 mM NaOH, and 1M sodium acetate in 100 mM NaOH. Injections were made every 40 min. All methods were based on protocols described by Hardy and Townsend (Hardy, M. R., and Townsend, R. R., "High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates", 1994, Methods Enzymol. 230: 208-225).